This Project tests the hypothesis that the initial steps in the interaction between the vector and the apical domain of well- differentiated (WD) airway epithelia constitute a principal barrier to efficient to efficient gene transfer. Because we hypothesize that both the vector binding and internalization steps are rate limiting, the project will focus on the cell biology of airway epithelia as it relates to these aspects of vector-cell interactions. Specific Aim 1 will define the barriers and targets in the apical domain of WD human airway epithelia. A combination of morphologic and immunohistochemical studies will quantitate the glycocalyleal components pertinent to gene transfer, the distribution of potential target receptors [seven transmembrane (7- TM) versus growth/trophic] on apical versus basolateral domains, the capacity for internalization of each membrane, and finally, the tight junctions, focussing on different airway regions (bronchial versus bronchiolar) within the lung. Specific Aim 2 will test the hypothesis that "modification of the host" to increase access of vectors to the basolateral domain of WD cells and/or basal cells will increase gene transfer efficiency. Two broad approaches are proposed: oxidant damage to epithelium, increasing permeability non-specifically; and cell biologic approaches to selectively increase the permeation of vectors through the tight junctions (TJ). Specific Aim 3 will test the hypothesis that vectors can be "modified" to target a class of receptor (the 7-TM) that are expressed in the apical membrane and internalized after agonist stimulation. Vectors will be directed to P2Y/2 receptors and other 7-TM receptors, using bis-specific and/or linked to modified natural ligands as the "cognate" moieties. For both "modifications of the host" and "modification of the vector" strategies, the proposed studies will assess both the efficiency and the safety aspects of each approach. For all studies, we will employ a spectrum of vectors, including AAV, lentiviral vectors, and adenoviral vectors. Complementary model systems will be employed, including the WD human air-liquid interface and the mouse nasal and tracheal models for in vivo studies. Our goal is to develop efficient gene transfer to WD airway epithelial cells in both the large and small airways of the CF lung.